Tooth organs form through the sequential reciprocal signaling interaction between ectodermally-derived epithelial cells and cranial neurocrest differentiated mesenchymal cells. Dental mesenchymal cells adjacent to the dental epithelium, differentiate into odontoblasts, which form dentin. The dental epithelial cells differentiate into ameloblasts that go through multiple stages of differentiation to orchestrate enamel formation. The enamel epithelium does not exist in adult teeth, and currently there are not readily available sources of dental epithelial cells. This limitation, along with a lack of knowledge on mechanisms guiding the formation of human enamel, impacts our capacity to develop strategies for tooth regeneration. We have successfully microdissected ameloblasts from human fetal tooth organs and have begun to characterize these cells for expression of enamel matrix proteins by qPCR. In parallel studies we have differentiated human embryonic stem cells (hESCs) into epithelial phenotype. We hypothesize that dental epithelial lineage cells can be generated from pluripotent human embryonic stem cells. In this application, we will build on our preliminary research to identify genes and niches required for differentiation of hESC-derived epithelial cells into human ameloblast lineage cells. These studies will take us closer to our ultimate goal of tooth regeneration. Our specific aims are follows: 1) To identify genes that differentially expressed by human ameloblast lineage cells as compared to hESC-derived epithelial cells. 2) To determine the role of the dental mesenchyme in inducing hESC-derived epithelial cells commitment to ameloblast lineage cells. In aim 1, we will use Human Whole Transcript Microarray analysis and qPCR to identify transcription factors and cell surface proteins that are significantly up-regulated in ameloblast lineage cells as compared to hESC-derived epithelial cells. In aim 2, we will investigate odontogenic reciprocal signaling interactions in promoting epithelial cell differentiation by using spatially reconstituted three- dimensional niches containing Pitx2 positive epithelial cells and dental mesenchymal cells. Our ultimate goal is to develop cell-based strategies to replace missing teeth in humans. In this R03 grant, we will move a step closer to this goal, focusing on engineering human dental epithelial lineage cells, which is a critical challenge in tooth regeneration. In addition, these studies of epithelial- mesenchymal interactions of cells in tooth organ, can be used as a model system for a broader understanding of the mechanisms involved in the organgenesis of other epithelial appendages. PUBLIC HEALTH RELEVANCE: Demand for bioengineered teeth is increasing. However, engineering human dental epithelial cells is a critical hurdle in addressing this demand. Our goal in this proposal is to differentiate human embryonic stem cells into dental epithelial cells. We hypothesize that human dental epithelial cells can be generated from pluripotent human embryonic stem cells if the appropriate niches are provided.